Purification and properties of rabbit-liver glycogen synthase

Killilea, S.Derek; Whelan, W. J.

doi:10.1021/bi00651a028

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“…Liver phosphorylase kinase is partially dependent on Ca2+ for activity and can be activated by cyclic-AMP-dependent protein kinase [I I]. Liver glycogen synthase is inactivated by both cyclic-AMP-dependent protein kinase [12,13] and a 'cyclic-AMP-independent' glycogen synthase kinase that has been partially purified from liver [13]. Liver glycogen synthase b has been reported to contain 12 mol alkali-labile phosphate/mol subunit [14], suggesting it may be even more highly phosphorylated than the muscle enzyme.…”

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“…Liver phosphorylase b is poorly activated by 5'AMP in comparison to the muscle enzyme while liver phosphorylase a is dimeric under conditions where the muscle enzyme is a tetramer [20]. Liver glycogen synthase is also dimeric [12,13], in contrast to muscle glycogen synthase which is a tetramer (reviewed in [?I).In the preceding paper [5] protein phosphatases were classified into one of two types, depending on whether they dephosphorylated the P-subunit of muscle phosphorylase kinase and were inhibited by inhibitor-I and inhibitor-2 (type-I protein phosphatases), or whether they dephosphorylated the r-subunit of phosphorylase kinase and were insensitive to inhibitor-I and inhibitor-2 (type-2 protein phosphatases). In this paper we have used the phosphorylated proteins of muscle glycogen metabolism to investigate the nature of the protein phosphatases that regulate glycogen metabolism in rabbit liver and rabbit skeletal muscle.…”

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The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

1983

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The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-I were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-I, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 232, 255-2611 were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors.Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCI), 2A1 (0.2 M NaCI) and 2Az (0.28 M NaCI) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-M,,,,,, catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2Az to more active 34000-Mr~,,,~ catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2Az possessed identical substrate specificities, preferentially dephosphorylated the r-subunit of phosphorylase kinase, were unaffected by inhibitor-I and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2Al and 2Az were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP.Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98 000. Protein phosphatase-2B was completely inhibited by 100 pM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B.Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2Al. These activities were completely resolved by gel filtration, since the Mr(,,,) of protein phosphatase-2C was 46000.Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg . ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2Al. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2.Most, if not all, of the protein phosphatases described in the literature as enzymes active on the phosphorylated proteins of glycogen metabolism can now b...

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The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

1983

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“…6) accordingly showed a band of M , 95000 (phosphorylase) as well as the synthase band at 85000, but only one very weak additional band at 73 000. A subunit M , of 85 000 corresponds to values of 85000-88000 reported for synthase I from rabbit muscle and liver [13,41].…”

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confidence: 89%

“…[7] and 3 -4 times lower than for synthase I isolated from rabbit muscle [42] and liver [41]. The assessment of the purity of the final glycogen-free enzyme was hampered by the necessity of adding albumin to the enzyme during lyophilization and further purification by gelfiltration on Sephadex G-200 resulted in low recovery.…”

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Purification and Properties of Glycogen Synthase I from Human Leukocytes

Sølling¹,

Esmann²

1977

European Journal of Biochemistry

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Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen-enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7 -11 U/mg protein, or 4-5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85 000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13 500 cal/mol (56.5 kJ/mol), corresponding to a Qlo of 2.2. In the presence of glucose 6-phosphate or Na2S04, the enzyme showed a broad pH optimum between pH 6.8-9.2. In the absence of these ligands and in particularly in the presence of Mg', the enzyme is sensitive to small changes of pH in the interval pH 7.4-8.4.During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing, During 24-h incubations, synthase I undergoes a spontaneous, temperature-dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6-phosphate, Na2S04, inorganic phosphate, UDP and glycogen. Mgz+ and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years.Glycogen synthase, in most vertebrate tissues, exists in a phosphorylated, physiologically inactive D form [1,2], and a non-phosphorylated I form, which, when present, is always active [2,3]. These enzyme forms are interconvertible by phosphorylation-dephosphorylation reactions, which are subject to hormonal control [1,3]. The existence of both forms of synthase, as well as the enzymes responsible for the interconversion reactions, have been demonstrated in human polymorphonuclear leukocytes in our laboratory [4-91. Recently a third enzyme form, synthase R, with rheostatic properties has been kinetically characterized [ 101.Synthase D has recently been found to follow a rapid-equilibrium random bi-bi mechanism [l 13. A similar exploration of the bisubstrate and modifier kinetics of synthase I has been hampered by the fact that the I enzyme is readily inactivated [12-141 and is also more susceptible to proteolytic degradation Enzyme. Glycogen synthase (EC 2.4.1.11).than the D enzyme [15]. In addition, glycogen, besides being a substrate, also activates synthase I in a timedependent and concentration-dependent manner, as will be described in a following communication [16].The present paper describe...

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Glycogen (starch) synthase

Springer Handbook of Enzymes

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Purification and properties of rabbit-liver glycogen synthase

Cited by 50 publications

References 42 publications

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

The Proteink Phosphatases Involved in Cellur Regulation. 2. Glycogen Metabolism

Purification and Properties of Glycogen Synthase I from Human Leukocytes

Glycogen (starch) synthase

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